Erythritol-producing moniliella strains

ABSTRACT

An isolated strain of the  Moniliella  species that converts glucose to erythritol with a conversion rate of at least about 45% is disclosed, as is a method of producing erythritol from such a strain.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional and claims the benefit of priorityunder 35 USC 120, of U.S. application Ser. No. 09/759,778, filed Jan.12, 2001 now U.S. Pat. No. 6,455,301. The disclosure of the priorapplication is considered part of, and is incorporated by reference in,the disclosure of this application.

BACKGROUND

Erythritol is a sugar alcohol that can be found in lichens, hemp leaves,and mushrooms. It is also savored in fermented foods such as wine, soyasauce, or saki (Sasaki, T. (1989) Production technology of erythritol.Nippon Nogeikagaku Kaishi 63: 1130-1132). Erythritol is a four-carbonpolyol, which possesses several properties such as sweetness (about70-80% of sucrose), tooth friendliness, very low calorific value (0.3kcal/g, a tenth of sucrose), non-carcinogenicity and, unlike otherpolyols, causes little, if any, gastro-intestinal discomfort (Harald andBruxelles (1993) Starch/Starke 45:400-405).

Traditional industrial erythritol production is carried out by addingcatalysts such as hydrogen and nickel to the raw material sugars underthe environment of high temperature and high pressure. Another processis performed by the chemo-reduction of raw materials such asmeso-tartarate (Kent, P. W., and Wood, K. R. (1964) J. Chem. Soc.2493-2497) or erythrose (Otey, F. H., and Sloan, J. W. (1961) Ind. Eng.Chem. 53:267) to obtain erythritol. In addition, erythritol can beproduced by a number of microorganisms. Such organisms include highosmophilic yeasts, e.g., Pichia, Candida, Torulopsis, Trigonopsis,Moniliella, Aureobasidium, and Trichosporon sp. (Onishi, H. (1967) HakkoKyokaish 25:495-506; Hajny et al. (1964) Appl. Microbiol. 12:240-246;Hattor, K., and Suziki, T. (1974) Agric. Biol. Chem. 38:1203-1208;Ishizuka, H., et al. (1989) J. Ferment. Bioeng. 68:310-314.)

SUMMARY

The invention features isolated strains of the Moniliella species withenhanced capacities for the conversion of glucose to erythritol. Suchstrains can produce erythritol from glucose with a conversion rate of atleast about 35%, 40%, 45%, 50%, 55%, 60%, 65% or greater under optimalconditions.

Strains of the invention include isolates of Moniliella from a naturalsource; and the mutants of a Moniliella strains, e.g., a Moniliellastains assigned the American Type Culture Collection (ATCC) accessionnumbers of PTA-1227, PTA-1228, PTA-1229, PTA-1230, and PTA-1232. Oneparticular mutant strain is the isolated strain, N61188-12, depositedwith the American Type Culture Collection with the accession numberPTA-2862.

As used herein, the term “mutant” refers to a strain whose geneticcomposition differs by at least one nucleotide, e.g., a substitution,insertion, or deletion, relative to a reference or parent strain. Amutant of the invention can be produced by a number of methods. Onemethod is the selection of strains with increased erythritol conversionrates relative to a parent strain. The strains can be obtained by randommutagenesis of the parent strain, e.g., by means of a chemical mutagen,a transposon, or irradiation. In addition, a mutant strain of theinvention can include a recombinant nucleic acid sequence. For example,a mutant may be a strain that harbors an additional nucleic acidsequence, e.g., a sequence transformed, transduced, or otherwiseinserted into a cell of the parent strain. The additional nucleic acidsequence can encode a polypeptide that is generally or conditionallyexpressed. Alternatively, the additional nucleic acid sequence canencode a nucleic acid sequence capable of altering cell physiology,e.g., an anti-sense, a ribozyme, or other nucleic acid sequence. Inanother instance, the inserted nucleic acid is inserted into anendogenous gene, and alters (e.g., enhances or disrupts) its function.For example, the inserted nucleic acid can be a knockout construct thatinactivates the endogenous gene; or an artificial enhancer or promoterthat increases transcription of the endogenous gene. The mutation candisrupt the ability of the parental strain to import, assimilate, orconsume erythritol or mannitol.

The invention also features a method of producing erythritol. The methodincludes growing a Moniliella strain of the invention, e.g., an enhancedmutant, in a culture; and purifying erythritol from the culture, e.g.,from the supernatant or from the cell pellet.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DETAILED DESCRIPTION

The fungus Moniliella is capable of fermenting simple sugars to produceerythritol, a well-relished component of many cuisines. Screening andmutagenesis are used to identify improved strains of Moniliella that arecapable of highly efficient erythritol production yields. Such strainsare ideal for large-scale erythritol production, as can be achieved bythe exemplary methods described herein.

Isolation of Enhanced Erythritol Producing Strains

Isolates of Moniliella can be obtained from a natural source asdescribed in U.S. patent application Ser. No. 09/585,926, filed Jun. 2,2000, now U.S. Pat. No. 6,300,107. For example, isolates of Moniliellacan be obtained natural sources having high sugar content include honey,preserved fruit, and pollen. Each strain is identified based on itscapability to convert glucose to erythritol and its variousmorphological and physiological traits. As used herein, the“glucose-to-crythritol conversion rate” is defined as the amount oferythritol produced divided by the amount of glucose consumed. Theresulting ratio can be expressed as a percentage. Theglucose-to-erythritol conversion rate of a fungal strain can becalculated by the following method. The strain is first cultured in a10-ml broth containing 30% glucose and 1% yeast extract (initial celldensity 1·10⁵ cells/ml) in a 50 ml flask in a rotary shaker at 150 rpmand 30° C. for 6 days. Then, both the concentration of erythritol in themedium and the concentration of glucose in the medium are determined.The conversion of 1 g of glucose into 0.3 g of erythritol is termed a30% conversion rate. The morphological traits are determined followinggrowth on 4% malt extract, 0.5% yeast extract agar for 10 days at 20° C.See The Yeasts, A Taxonomic Study, Edited by Kurtzman et al., 4th Ed.,page 785, Elsevier, Amsterdam (1998).

A mutant of a Moniliella strain can be obtained by the mutagenesismethod described in Ishizuka, et al. (1989) J. Ferment. Bioeng.68:310-314, or a variation thereof (see also U.S. Pat. No. 5,036,011).One variation for the mutagenesis of Moniliella cells withN-methyl-N-nitrosoguanidine (NTG) is described as follows. Moniliellacells are inoculated in broth with 30% glucose and 1% yeast extract, andcultured overnight at 30° C. on a rotary shaker at 150 rpm. This cultureis diluted 1:100 into 10 ml of broth with 30% glucose, and incubated at30° C. on a rotary shaker at 150 rpm for 1 day. The culture broth iscentrifuged at 3,000 rpm for 15 min to form a cell pellet and thesupernatant is discarded. The cell pellet is washed with 10 ml ofsterile 0.1 M pH 7.0 phosphate buffered saline (PBS). The suspension iscentrifuged (3,000 rpm, 15 min) and the supernatant is again discarded.The cells are resuspended in PBS, with 150 μg/ml NTG for 10 minutes.

After treatment with NTG, the Moniliella cells are grown in a glucosesolution for 3 hours. The culture is then diluted appropriately andspread onto the medium containing 65% glucose and incubated at 30° C.for 6 days. Colonies are selected randomly, inoculated into brothcontaining 30% glucose, and incubated at 30° C. on a rotary shaker at150 rpm overnight. A 1:100 dilution of the overnight culture is used toinoculate into a 30% glucose solution (10 ml) that is incubated at 30°C. on a rotary shaker at 150 rpm for 4 days. The medium from thisculture is then centrifuged at 12,000 rpm for 10 min. The supernatant isdiluted appropriately and the amount of residual glucose is measuredusing the DNS method (see below). Cultures with higher glucoseconsumption (i.e., lower residual glucose) are further analyzed todetermine erythritol yield. The HPLC method described below can be usedto quantitate erythritol yield. Cultures with indications of elevatederythritol yield are subject to further verification. For example,individual colonies are obtained for the culture, re-grown as describedabove, and reanalyzed. Selected colonies can be improved by additionalrounds of mutagenesis according to these procedures.

Measurement of Residual Glucose

4-day-old culture broth is collected and centrifuged at 12,000 rpm for10 min. The supernatant is diluted appropriately. 1 ml of each dilutedsolution is added to 0.5 ml of DNS (dinitrosalicylic acid) reagent. DNSreagents (e.g., a. 1% 3,5-dinitrosalicylic acid (DNS). b. 0.2% phenol;c. 0.05% NaHSO₃ or 0.025% Na₂S₂O₃; d. 1% NaOH; e. 0.5% potassium sodiumtartrate tetrahydrate) were prepared and used according to methoddescribed in Miller, G. L. (1958) Anal. Chem. 31:426-428. The mixture ismixed well and incubated at 100° C. for 5 min. After cooling under roomtemperature, 9 ml water is added and the absorbance at 540 nm(OD_(540 nm)) is determined. The absorbance at 540 nm is used todetermine the concentration of glucose by comparison with the standardcurve, obtained by measuring pure glucose at various concentrations.

Measurement of Erythritol Concentration

The amount of erythritol in a supernatant can be quantitated by HPLC andTLC, e.g., to determine the erythritol-producing capacity of a strain.HPLC analysis is performed by Hewlett Packard H4033A analyzer on anIon-300 chromatography column, using 0.1 N sulfuric acid as the flowingphase with a flowing rate of 0.4 ml/min, the temperature being set at75° C. For TLC analysis, the Neissner et al. procedure is followed.(Neissner, et al. 1980. Herstellung, aanalyse und DC-trennung vonfettsaure erythritpartialestem. FETTE SEIFEN ANSTRICHMITTEL. 82:10-16.).After rinsing Kieselgel 60F254 (Merck) with 4% boric acid, the gel isheated in an incubator at 105° C. for 20 minutes before use. Thespreading solvent is ethylmethylketone:acetone:water (100:10:10 by vol.)and the color-developing agent is KMnO₄ in concentrated sulfuric acid.

Erythritol purified from a supernatant by HPLC or TLC can be furtherpurified by extraction and then dried under reduced pressure. Thefurther purified product and an erythritol standard are acetylatedaccording to the method of Shindou et al. (Shindou et al. 1989. J.Agric. Food Chem. 37:1474-1476.). Erythritol standards are commerciallyavailable, e.g., from Merck, Germany. The resulting sample can beassayed by GC-MS to determine if the re-purified product was identicalto that of the standard sample.

Large Scale Production of Erythritol

Following the specific examples provided below, a skilled artisan canoptimize erythritol yield of a mutant Moniliella strain by identifyingpreferred pH, temperature, and carbon source for growth andfermentation. Similar analysis can be used to optimize aeration,stirring speed, culture volume, and culture time.

To produce erythritol on a larger scale, 0.2 ml of Moniliella cellspreserved in glycerol are added to 50 ml of broth in a 500 ml flask, andincubated at 30° C. on a rotary shaker at 150 rpm for about 24 hours.From this culture, 2 ml are used to inoculate a second 500 ml flask with50 ml of broth. The second culture is incubated at 30° C. on a rotaryshaker at 150 rpm for 48 hours. The second culture broth is used toinoculate 2 L of broth in a 5 L fermentor (NBS. Edison, N.J., USA). Theculture conditions are as follows. Aeration: 1 VVM; stirred speed: 500rpm; temperature: 30° C.; culture period: 5-7 days.

For these purposes, the broth can consist of 30%, 35%, 40%, 45%, or 50%glucose, together and 1% yeast extract. In addition, KM72 and KM72F(Shin Etsu, Shin-Etsu Chemical Co., Ltd. 6-1, Ohtemachi 2-chome,Chiyoda-ku, Tokyo, Japan) can be used as a defoamer.

Purification of Erythritol

Media from the fermentor is centrifuged to separate the culturesupernatant from pelleted cells. The supernatant is decolored by passageover active carbon (e.g., powdered carbon as can be obtained from alocal supplier). The decolored supernatant is desalted andde-proteinated by consecutive passage of over a cation exchange resin,DIAION, WA30 (Mitsubishi) and an anion exchange resin, AMBERLITE IR120NA (Rohm and Haas Company). The resulting solution is concentrated withthe following apparati: EYELA Rotary Vacuum Evaporator N-N Series; EYELAWaterbath SB-450; and EYELA, Aspirator A-3 (Tokyo Rikakikai Co. LTD).The concentrated solution is crystallized at room temperature. Crystalsare optionally washed with or re-crystallized in hydrous alcohol andwater (e.g., at 4° C.) to remove the trace impurities.

Verification of Erythritol Purification

To confirm the chemical identity of the purified product, the NMRspectra of the purified product is compared to the NMR spectra of astandard, e.g., erythritol purchased from Merck Co. (NJ, USA), oranother commercial supplier. The samples are dissolved in 100% D₂O andplaced in an NMR spectrometer (Bruker AM-500, Germany). The followingconditions are used for ¹H NMR spectra: 400.135 MHz; pulse length: 4.0μs; acquisition time: 1.245 sec; pulse delay: 1 sec; chemical shifts:D₂O as 0 ppm. The following conditions are used for ¹³C NMR spectra:100.536 MHz; pulse length: 5.0 μs; acquisition time: 0.623 sec; pulsedelay: 2 sec; chemical shifts: 10 mM DSS as 0 ppm.

A skilled artisan can obtain a fungal mutant of the invention andutilize it to the fullest extent to produce erythritol based on theguidance of the following specific example, which is merelyillustrative, and not limitative of the scope of the invention. Allpublications cited herein are incorporated in their entirety byreference.

EXAMPLE

Moniliella Mutant Isolation

The erythritol-producing fungi Moniliella PTA-1230 was mutagenized withNTG by the method described above. The procedure was repeated such thatan improved erythritol producer isolated in one round is used as theparent strain for the subsequent round. The N61188-12 mutant strain(ATCC deposit PTA-2862) was isolated afler six rounds of mutagenesis.

The N61188-12 mutant strain and the parental PTA-1230 were cultured inbroth containing 35% glucose and 1% yeast extract on rotary shaker at150 rpm for 6 days at the temperature of 25° C., 30° C., 34° C., and 37°C. At each of these temperatures, the glucose-to-erythritol conversionrates were respectively: 43.9%, 61.4%, 17.8%, and 2.2%, for theN61188-12 mutant strain; and 18.9%, 30.5%, 17.9%, and 7.7% for theparental PTA-1230. At 25° C. and 30° C., the erythritol yields of theN61188-12 strain were at least twice as great as that of the PTA-1230.The 61.4% yield observed for the N61188-12 strain was unexpected, as itis remarkably close to the theoretical upper limit for completeconversion of glucose to erythritol −68%.

For the purposes of verification, pure erythritol was obtained from afermentor culture of the N61188-12 strain using the above-describedmethods. The pure erythritol from N61188-12 was analyzed by nuclearmagnetic resonance as described above. Its spectra were identical to thespectra of an erythritol standard indicating that the product recovered,purified, and crystallized was, indeed, erythritol.

Optimization of Erythritol Production Conditions.

The erythritol yields were determined in parallel for the parentalPTA-1230 and the N61188-12 strain under conditions of varying pH,temperature (see above), and carbon source.

pH. The parental PTA-1230 and the N61188-12 strains were cultured in 35%glucose and 1% yeast extract broth adjusted to various pH's at 30° C. ona rotary shaker at 150 rpm for 6 days. For the pH's 3.0, 4.0, 5.0, 6.0,and 7.0, the erythritol yield of the PTA-1230 was 31.2%, 39.3%, 38.4%,34.4%, and 34.2% respectively, whereas the erythritol yield of theN61188-12 strain was 56.6%, 59.4%, 58.5%, 60.3%, and 57.3%,respectively.

Glucose concentration. Culture broths containing 20%, 30%, 35%, 40%, and50% glucose together with 1% yeast extract were prepared. Both PTA-1230and N61188-12 strains were cultured in the above broths at 30° C. on arotary shaker at 150 rpm for 6 days. At each of these glucoseconcentrations, the erythritol yield of the PTA-1230 strain was 40.6%,37.1%, 34.5%, 29.4%, and 19.2%, respectively, whereas the erythritolyield of the N61188-12 strain was 56.3%, 57.5%, 62.8%, 55.6%, and 35.8%,respectively (Table 10). The optimal yield of the PTA-1230 strain waswith the 20% glucose solution, the yield decreasing with the increasingglucose concentration. The optimal yield of the N61188-12 strain waswith the 35% glucose broth. The yields obtained from other glucoseconcentrations, such as 20%, 30%, and 40% glucose solution, were similarto each other, while that obtained from 50% glucose solution was reducedto 35.8%. At high glucose concentrations, e.g., 40% and 50%, theerythritol yield of the N61188-12 strain was nearly twice that of thePTA-1230 strain. These results indicate the unexpectedly improvederythritol production capacity of the N61188-12 strain in comparison tothe wild type PTA-1230 strain.

Carbon Source. The culture broths containing 35% of either glucose,maltodextrin, maltose, sucrose, fructose, or lactose as carbon source,and 1% yeast extract as nitrogen source were prepared. Both PTA-1230 andN61188-12 strains were cultured in above broths at 30° C. on a rotaryshaker at 150 rpm for 6 days. Respectively for glucose, maltodextrin,maltose, sucrose, fructose, or lactose, strain PTA-1230 produced 120.8,44.5, 0, 154.0, 111.0, and 0 g/L of erythritol, whereas the N61188-12strain produced 220.0, 15.1, 22.8, 239.4, 211.4, and 0 g/L. Theseresults indicated that sucrose has best conversion capacity for bothfungal strains, and the next being glucose, and then fructose. Notably,strain PTA-1230 cannot utilize maltose and lactose for erythritolproduction, whereas the N61188-12 strain can utilize maltose, but notlactose for erythritol production. For the PTA-1230 strain, theerythritol yield using sucrose as the carbon source was 27.5% higherthan that using glucose, whereas the yield was only 9% higher under thesame conditions for the N61188-12 strain.

Byproduct Accumulation. The concentrations of the metabolicbyproducts—glycerol, pentitol, and alcohol—were monitored in theaforementioned carbon sources. For example, when glucose was used as thecarbon source, the concentration of glycerol and pentitol in thePTA-1230 strain culture broth was 36.4 and 17.2 g/L, respectively,whereas there was no glycerol present, and the content of pentitol wasonly 3.8 g/L in the N61188-12 strain culture broth. Results foradditional carbon sources are illustrated in Table 1.

No alcohol was producing during the fermentation of the PTA-1 230 strainwith glucose as the carbon source. However, in other carbon sources,both the PTA-1230 and N61188-12 strains produced alcohol for the initialfive days after inoculation. However, the alcohol was exhausted on the6^(th) day. The only exception was some residual alcohol (0.7 g/L) onthe 6^(th) day when fructose was used for the N61188-12 culture. In sum,these results indicate that the use of sucrose for culturing theN61188-12 strain results in a high conversion capacity to erythritolwithout the accumulation of byproducts.

TABLE 1 Production of erythritol and byproducts of PTA-1230 andN61188-12 strains Byproducts from PTA-1230 strain (g/L ) Byproducts fromN61188-12 strain (g/L) Carbon Source Erythritol Glycerol PentitolAlcohol Erythritol Glycerol Pentitol Alcohol glucose 120.8 36.4 17.2 0220 0 3.8 0 maltodextrin 45.5 0 0 0 15.1 0 0 0 maltose 0 0 0 0 22.8 0 00 sucrose 154 23.4 0 0 239.4 0 0 0 fructose 111 26.3 7.6 0 211.4 13.64.2 0.7 Each carbon source was present at 35%; nitrogen source 1% yeastextract; and incubation at 150 rpm, 6 days.

Gross properties of mutant strain N61188-12. The parental PTA-1230strain and the mutant N61188-12 strain were grown under variousconditions and compared. Their cell morphologies were substantially thesame. However, on plates, the mutant strain grew to only a quarter thesize of the PTA-1230 strain. Notably, the two strains have differentphysiological properties. These differences are reflected in theirabilities to ferment and assimilate different sugars. The mutantN61188-12 strain can ferment galactose (Table 2), whereas the PTA-1230strain cannot. In addition, the N61188-12 strain is unable to assimilateerythritol and mannitol (Table 3) in contrast to the PTA-1230 strain.

TABLE 2 Fermentation of various carbon sources Carbon source PTA-1230strain N61188-12 strain glucose + + galactose − + maltose + +sucrose + + lactose − −

TABLE 3 Assimilation study of PTA-1230 and N61188-12 strains on variouscarbon sources PTA-1230 Carbon source strain N61188-12 strainglucose + + galactose − − sorbose − − glucosamine − − ribose − − xylose− − L-arabinose − − D-arabinose − − rhamose − − sucrose + + maltose + +trehalose − − methyl-D-glucoside − − cellobiose + + salicin − −arbutin + + melibiose − − lactose − − raffinose − − melezitose − −inulin − − glycerol + + erythritol + W* ribitol − − xylitol − −arabinitol − − glucitol − − mannitol + − galactitol − − myo-inositol − −glucono-1,5-lactone + − 2-keto-gluconate − − gluconate − − glucuronate −− galacturona − − lactate − − succinate + + citrate − − methanol + +ethanol − − propane − − butane − − quinate − − saccarate − − galactonate− − W* refers to weak growth and meager assimilation of the carbonsource.

Cell Density. Under various conditions such as temperature, pH, carbonsource, and glucose concentration, the turbidity (A₆₆₀) of the culturebroth for the N61188-12 strain was less than that of the PTA-1230strain. Overall (except for use of maltose and lactose as the carbonsource), the turbidity of the culture broth for the N61188-12 strain wasbetween 31% and 77% of that for the PTA-1230 strain. In most cases theturbidity of the N61188-12 strain was less than 50% of the PTA-1230strain. Thus, it is inferred that the N61188-12 strain reduced theproportion of carbon source applied to cell growth, and insteadconverted a greater proportion of the carbon source into erythritol.

Other Embodiments

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A method of producing erythritol, the method comprising: growing astrain of the Moniliella species in a culture, wherein the strainconverts glucose to erythritol with a conversion rate of at least about45%; and purifying erythritol from the culture.
 2. The method of claim1, wherein the strain converts glucose to erythritol with a conversionrate of at least about 50%.
 3. The method of claim 1, wherein the strainconverts glucose to erythritol with a conversion rate of at least about60%.
 4. The method of claim 1, wherein the strain is a mutant of theMoniliella strain PTA-1227, PTA-1228, PTA-1229, PTA-1230, or PTA-1232.5. The method of claim 4, wherein the strain is a mutant of theMoniliella strain PTA-1227.
 6. The method of claim 4, wherein the strainis a mutant of the Moniliella strain PTA-1228.
 7. The method of claim 4,wherein the strain is a mutant of the Moniliella strain PTA-1229.
 8. Themethod of claim 4, wherein the strain is a mutant of the Moniliellastrain PTA-1230.
 9. The method of claim 4, wherein the strain is amutant of the Moniliella strain PTA-1232.
 10. The method of claim 8,wherein the strain is N61188-12, deposited with the American TypeCulture Collection with the accession number PTA-2862.